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Increase in detection of Corynebacterium diphtheriae in Canada: 2006–2019

KA Bernard1,2*, AL Pacheco1, T Burdz1, D Wiebe1 This work is licensed under a Creative Commons Attribution 4.0 International License. Abstract

Background: Increasingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been used to provide rapid, inexpensive and Affiliations precise identification of bacteria, includingCorynebacterium species. Only three Corynebacterium species are able to produce diphtheria toxin (DT), and strains recovered 1 National Microbiology may be either toxin-producing or non-toxin-producing. It appears the more precise bacterial Laboratory, Public Health Agency of Canada, Winnipeg, MB identification provided by MALDI-TOF systems has led to an increase in requests submitted to 2 the National Microbiology Laboratory (NML) for toxin testing. Department of Medical Microbiology, University of Manitoba, Winnipeg, MB Objective: To describe the number of isolates identified asC. diphtheriae, C. ulcerans and C. pseudotuberculosis, submitted to the NML between January 2006 and July 30, 2019, including their geographic area, source, and whether they produce DT. *Correspondence: [email protected] Methods: Referrals to the NML of human or animal isolates that were identified as any of those three Corynebacterium species were studied with respect to province, source and toxigenicity. Species identification was confirmed and then specimens were tested by polymerase chain reaction for the presence of tox genes and, if positive, for expression of DT by the modified Elek method. Analysis was descriptive.

Results: Over the study period, 639 isolates were identified asC. diphtheriae, 22 isolates as C. ulcerans; no isolates were identified asC. pseudotuberculosis. There was an increase in C. diphtheriae referrals for DT testing: from eight per year in 2006 to an average of 15 per month in 2019, or a 1,200% increase over the 13.6-year period. The referrals were primarily from western Canada (n=609/639; 95%). Most (638/639, 99%) were human isolates and most were obtained from cutaneous sites. Of those isolates, 87/639 (13.6%) were found to be toxigenic and 552/639 (86.4%) non-toxigenic. Among C. ulcerans referrals, 17/22 (77%) were from humans and five (23%) were from animals, with 10/22 (45%) being toxigenic.

Conclusion: There has been a marked increase in referrals to the NML for DT testing of Corynebacterium species. This could be due to the enhanced ability to identify these bacteria using MALDI-TOF systems. Ongoing monitoring will help to assess whether the increase is due solely to increased precision of diagnosis or whether these are emerging cutaneous pathogens.

Suggested citation: Bernard KA, Pacheco AL, Burdz T, Wiebe D. Increase in detection of Corynebacterium diphtheriae in Canada: 2006–2019. Can Commun Dis Rep 2019;45(11):296-301. https://doi.org/10.14745/ccdr.v45i11a04 Keywords: Corynebacterium diphtheriae, diphtheria-toxin-producing species, increase in recovery, MALDI-TOF Introduction

Classic Corynebacterium diphtheriae (C. diphtheriae) infection— In the past, Corynebacterium species isolated from causing sore throat, fever and respiratory symptoms—is rare in non‑respiratory clinical specimens were understudied and Canada (1–3). Since vaccination against diphtheria toxin (DT) is underreported. These bacteria were nearly always considered now part of the routine immunization schedule, there are fewer as contaminants, and were neither identified (i.e. genus and than four cases of this notifiable disease annually (4,5). species) nor tested for antimicrobial susceptibility, even if recovered in pure culture from sterile body fluids or deep

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wound abscesses (3). As well, unambiguous identification of systems can only identify these bacteria and cannot determine strains to genus and species could be difficult and expensive to whether or not any of these Corynebacterium species actually perform solely using phenotypic test methods (1,3). produce DT. Over the past few years, the National Microbiology Laboratory (NML) began to receive an increasing number of It is now more widely appreciated that non-respiratory requests for DT testing from public health laboratories and C. diphtheriae can cause skin and wound infections. Cutaneous veterinary laboratories across the country. The objective of diphtheria often presents as well-demarcated, sometimes this study was to describe the number and features of the foul-smelling ulcers or as nodules that are slow healing and C. diphtheriae, C.ulcerans and C. pseudotuberculosis samples highly contagious. People who are particularly at risk include submitted to the NML between January 2006 and July 30, 2019, those with co-morbidities such as HIV, hepatitis B or C, diabetes, including the percentage that produced DT. a history of recurrent ulcers, alcohol abuse, a history of intravenous drug use, living in poorer-socioeconomic conditions, homeless shelters or refugee camps, or who travelled to Methods countries where these pathogens are endemic (6–11). Treatment for cutaneous diphtheria often includes the use of the same Record review systemic antibiotics as those used for respiratory disease, plus The NML’s Special Bacteriology Unit (SBU) laboratory records the implementation of isolation precautions (6–10) to prevent were reviewed from 2006 to July 30, 2019 for test requests for bacteria from lesions serving as a reservoir for further spread of DT detection. Information reviewed here included province, infection (11,12). However, in some instances, difficult-to-treat site of infection if known, the species of DT-producing multidrug resistant strains of C. diphtheriae have been detected Corynebacterium involved and whether or not the strain was from cases of cutaneous diphtheria (13). Occasionally, sepsis and toxigenic. Referrals received from January to July 2019 were death may result. The role of prior vaccination with diphtheria further analyzed with respect to the source of the strain, as toxoid in preventing cutaneous infections is not well understood. these data were typical of referrals in other years. Information The use of antitoxin is generally not recommended, unless signs regarding vaccination history and underlying patient history was of systemic toxicity are present (7). generally scant or not provided.

Identification of the genus and species ofCorynebacterium Overview of specimen types received species has now been simplified by the widespread use of Since 2011, Cadham Provincial Laboratory (Manitoba’s public matrix-assisted laser desorption/ionization time-of-flight mass health laboratory) only forwards strains to the NML that have spectrometry (MALDI-TOF) instruments and more precise been found to be positive for tox gene targets by polymerase molecular methods. Proteins are extracted from bacteria and chain reaction (PCR) (D. Alexander, personal communication, the resulting fingerprints are then compared to entries housed July 24, 2019). Other provincial centres forward all strains that in the system’s data library. High confidence identification of may require testing for DT to the NML, as toxin analyses are species can be obtained within a few minutes, at a cost of not offered in their own laboratory settings. The NML receives less than $1.00 Canadian per sample (14,15). The MALDI‑TOF pure cultures of bacterial isolates that have been recovered at procedure has made it possible to routinely identify the provincial public health laboratories or by their clientele, which diphtheria-toxin-producing species: C. diphtheriae; C. ulcerans; the provincial public health laboratories then forward to the NML and C. pseudotuberculosis. Infections involving C. ulcerans and for toxin testing. Occasionally strains are received from veterinary C. pseudotuberculosis are rare in humans and are acquired laboratories. through infected animals. The C. ulcerans infections in humans have been linked to contact with diseased pets, such as dogs or Confirmation ofCorynebacterium diphtheriae cats, whereas C. pseudotuberculosis occurs in sheep and goats, isolates so veterinarians or animal handlers are at increased risk for acquisition (3). From 2006 to approximately 2018, SBU strains had been tested phenotypically using an API CORYNE (bioMérieux, In Canada, the use of MALDI-TOF instruments began about France) panel to provide species-level identification and to 2012. These instruments are now widely distributed across the assist with categorizing the isolate with respect to one of country—in all provincial public health laboratories, in many the four C. diphtheriae biovars (i.e. biotypes: gravis; mitis; private/public hospital institutions and in veterinary laboratories belfanti; and intermedius) (1,3). The API CORYNE panel specializing in infectious diseases of animals. The clear benefits unambiguously identifiedC. diphtheriae for strains previously for routine use of MALDI-TOF systems are the significantly characterized by the gold standard 16S rRNA gene sequencing reduced costs for characterizing bacteria and the greatly method; molecular methods are otherwise used at the NML to improved speed at which pathogens such as C. diphtheriae can corroborate species level identification as required. The NML be identified. These benefits result in prompt implementation ceased characterization of strains to biovar as of May 2019, of appropriate treatment options. However, MALDI-TOF except upon request. Corynebacterium diphtheriae identification

Page 297 CCDR • November 7, 2019 • Volume 45–11 SURVEILLANCE was validated for use by the NML for the Bruker Microflex and increased to 115. Between January and July 2019, an average Biotyper library, where this method also provides unambiguous of 15–16 referrals per month had been received at the identification of species (data not shown). NML: extrapolated, this would be approximately 185 by the end of the year. Comparing the pre-MALDI-TOF era for toxin testing Identification ofCorynebacterium ulcerans and of eight strains (in 2006) to an estimated 185 referrals projected C. pseudotuberculosis isolates for 2019, this represented a 1,200% or 12-fold increase over the review period (Figure 1). These species are readily discernable from all others in the genus Corynebacterium, including C. diphtheriae, but are otherwise Figure 1: Corynebacterium diphtheriae referrals for not easily differentiated from each other by commonly-used toxin testing by year, subset by number of toxigenic laboratory methods (1). Phenotypic results for these species, strains using conventional sugars or API CORYNE panels, are very 120 120 112 similar (1,2). These species have 99.4% similarity to each other 105 100 90 by 16S rRNA gene sequencing, which is too close to provide 2012–2013 MALDI starts 80 to become more definitive discrimination from each other, and can produce widely used in Canada 65 60 similar scores to each other using MALDI-TOF systems (data 40 33 not shown) (1). Therefore, precise identification of these 22 21 24 17 15 20 12 11 11 13 species is corroborated at the NML by use of partial rpoB gene 8 7 8 9 10 0 3 3 3 2 0 1 1

Referrals for toxin testing 0 sequencing (16). 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Year Detection of diphtheria tox gene targets and C. diphtheriae received (n) C. diphtheriae toxigenic (n) Abbreviations: C. diphtheriae, Corynebacterium diphtheriae; MALDI, matrix-assisted laser expression of diphtheria toxin desorption/ionization spectrometry

The SBU had used conventional PCR approaches to detect tox gene targets (13), but switched to a real time PCR-based Strains identified as C. diphtheriae were derived primarily method in 2018. Real-time PCR detects the tox gene and from western Canada (n=609/639; 95%), that is, from British a region of the rpoB gene specific forC. diphtheriae using Columbia, Alberta, Saskatchewan and Manitoba, with small a Quant Studio platform (Applied Biosystems) (17,18). The numbers from Ontario, Quebec and New Brunswick and from toxin‑producing strain, C. diphtheriae ATCC 19409 (NCTC outside of Canada (Figure 2). 3984), and the non‑toxigenic strain, C. xerosis ATCC 373T, were used as positive and negative controls, respectively. Expression Figure 2: Corynebacterium diphtheriae and C. ulcerans of DT was detected by performing the modified Elek test only referrals, by province for strains where tox genes had been detected by PCR (19,20).

Elek‑positive results may be reported at 48 hours, or as soon 400 375 as expression is visually detected, with negative results being 350 reportable after 48 hours. Isolates that are found to be positive 300 for tox gene by PCR but negative for DT expression by Elek are 250 reported as non-toxigenic (20); these occur rarely in Canada, 200 accounting for 8% or fewer of all tox gene-positive strains (21). 134 150

100

Referrals for toxin testing 65 35 50 Results 9 13 2 0 1 0 6 4 3 0 0 5 8 1 0 CB AB SK MB ON QC NB IPE Autre a Over the study period there were a total of 639 isolates Provinces identified asC. diphtheriae, where 638/639 (99%) were from C. diphtheriae referrals (n) human disease and 1/639 (1%) was from a horse. Twenty-two C. ulcerans referrals (n) isolates (n=17; 77% from humans and n=5; 23% from animals) Abbreviations: AB, Alberta; BC, British Columbia; C., Corynebacterium; MB, Manitoba; NB, New Brunswick; ON, Ontario; PEI, Prince Edward Island; QC, Quebec; SK, Saskatchewan were identified asC. ulcerans and no isolates were identified as a Other, sources outside of Canada C. pseudotuberculosis.

Corynebacterium diphtheriae Referrals of C. diphtheriae subdivided by province were as Between 2006 and 2012, an average of fourteen isolates of follows (% of n=631): British Columbia, 375 (59%); Alberta, 134 C. diphtheriae was referred annually. Between 2013 and 2015 (21%); Saskatchewan, 35 (5%); Manitoba, 65 (10%); Ontario, (which coincided with the increasing use of MALDI-TOF), the 13 (2%); Quebec, six (0.9%); and New Brunswick, three (0.4%). number of annual referrals increased to an average of 40. No C. diphtheriae referrals were received from Nova Scotia, Between 2016 and 2018, the number of annual referrals Prince Edward Island or Newfoundland, and eight were received

CCDR • November 7, 2019 • Volume 45–11 Page 298 SURVEILLANCE from outside of Canada. Referrals of C. ulcerans by province less than 15 years. These are predominately non-toxigenic were as follows (% of n=21): British Columbia, nine (43%); C. diphtheriae strains from cutaneous sources in humans Alberta, two (9%); Manitoba, one (5%); Quebec, four (19%); and recovered in pure culture or co-recovered in association with Prince Edward Island, five (24%, all from animals). No strains other Gram-positive bacteria (6). Although the overall numbers were referred from Saskatchewan, Ontario, New Brunswick, were small, a 35% increase of referrals involving C. ulcerans was Nova Scotia or Newfoundland, and one from outside of Canada. found, that included strains from humans and animals. Similar increases of both C. diphtheriae and C. ulcerans have been The majority of the 105 C. diphtheriae strains from 2019 were reported in other countries including the United States (7–11). obtained from skin infections. There were 89 (85%) specimens This increase is likely due in part to the increasingly widespread from cutaneous sites that included the abdomen and use of MALDI-TOF systems and precise molecular identification abscesses/wounds from the ankle, arm, elbow, foot, finger, methods. It may also reflect thatC. diphtheriae is an emerging head/scalp, hip, leg, knee, shin, thigh, thumb, tibia and toe; pathogen for non-respiratory infections. 16 specimens (15%) from non-cutaneous sites that included blood culture (n=1), uterine (n=1), ear (n=2), sinus, sinus washing, Clinical implications nasal wound and nose (n=5), throat (n=4), sputum (n=1) and Canadian clinicians should be aware that identification of unknown sites (n=2). C. diphtheriae and C. ulcerans from certain patient and specimen types is becoming increasingly common. Strains should be tested In 2019, there were 10 toxigenic strains recovered from nose for toxin production and, if toxigenic, a public health response, or cutaneous sites. These referrals were from Alberta (n=8), including testing/monitoring of contacts of patients, may be British Columbia (n=1) and Manitoba (n=1). No patient was indicated in addition to the usual treatment approaches (5). symptomatic of classic, respiratory-type diphtheria. Antibiotic sensitivity testing of these species has only rarely been Corynebacterium ulcerans requested but may be useful. In a recent article, we described A small number of referrals (n=22, Figure 3) were identified as antimicrobial susceptibility testing (AST) results based on an C. ulcerans. Seventeen (77%) samples were obtained from human ancillary study of 195 strains of C. diphtheriae and 20 strains of cases, with sources described as pus or from cutaneous sources C. ulcerans (22). Although these species are usually susceptible (arm, elbow, foot, leg or toe wounds). Five animal‑derived to first line antibiotics (22), the occasional strain ofC. diphtheriae specimens were also received from mink (n=1, lung), dog has been found to be penicillin or multidrug resistant (13,23); (n=1, ear), cat (n=1) and horse (n=2, abscess, skin). Such testing in some instances, azithromycin may be indicated (10). Of had also increased by approximately 35% between 2006–2012 note, the Canadian notifiable disease definition for diphtheria and 2013–2019 (Figure 3). Critically, although numbers were includes C. diphtheriae as well as both C. ulcerans and small, we found that 10/22 (45%) of C. ulcerans strains produced C. pseudotuberculosis and, if identified in a symptomatic patient, DT. would be classified as a confirmed case (5). Cutaneous diphtheria would also be classified as a confirmed case if a toxigenic strain Figure 3: Corynebacterium ulcerans referrals for toxin was recovered from a wound/cutaneous site (5). testing by year, subset by number of toxigenic strains

5 5 Limitations 5.0 4.5 4 4 There are some inherent limitations to the results shown here. It 4.0 is not mandatory for laboratories to send these taxa to the NML 3.5 3 2012–2013 MALDI starts to 3.0 become more widely used in Canada for further characterization. For example, Cadham Provincial 2.5 2 2 2.0 Laboratory conducted their own DT testing by PCR on more than 1.5 1 1 1 1 1 1 1 600 strains of C. diphtheriae in the first six months of 2019— 1.0

Referrals for toxin testing 0.5 all were found to be negative for the presence of tox genes 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.0 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 (D Alexander, personal communication, July 24, 2019). These

Year were not referred to the NML so the data presented here from

C. ulcerans received (n) C. ulcerans toxigenic (n) the NML is an underestimate.

Abbreviations: C. ulcerans, Corynebacterium ulcerans; MALDI, matrix-assisted laser desorption/ionization spectrometry Identification ofC. diphtheriae or C. ulcerans from a variety Notes: Five C. ulcerans strains shown from 2019 were received from a veterinary laboratory. The remaining 17 samples were obtained from human sources of clinical specimens, especially cutaneous sites, requires that local laboratories are able to select for/recognize these agents from among flora found in the biomes of infected cutaneous or respiratory sites. An algorithm may assist in the process of Discussion determining putative C. diphtheriae or C. ulcerans strains (1). Furthermore, it is difficult to conjecture if the lack of strains The NML has documented a 1,200% or 12-fold increase in received for DT testing from central and eastern Canada reflects referrals of Corynebacterium specimens for DT testing in that no actual cases exist, or they occur more rarely than in the

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Funding west. It may reflect the use of different guidance documents or different laboratory practices. Public Health Agency of Canada A Base funding was used for this study. Future research Ideally, the next steps could include both clinical and laboratory research, which would include assessment of cutaneous References diphtheria as an emerging infection and collection of more 1. Bernard KA. Coryneform Gram-Positive Rods. In: Carroll complete clinical data on treatment versus outcome. Further KC, Pfaller MA, Landry ML, McAdam AJ, Patel R, Richter laboratory research on these agents includes consensus-based SS, et al., editors. Manual of Clinical Microbiology. 12th ed. development of a national and internationally-based typing Washington, DC: ASM Press; 2019. p. 488-534. scheme for characterization of such strains at whole genome 2. Bernard KA, Funke G. Genus I. Corynebacterium Lehmann sequence level, for use in silico tracking of strains and for better and Neumann 1896, 350AL emend. Bernard, Wiebe, Burdz, characterization of virulence factors. Reimer, Ng, Singh, Schindle and Pacheco 2010, 877. In: Whitman WB, Goodfellow M, Kämpfer P, Busse H-, Trujillo Conclusion ME, Ludwig W, et al, editors. Bergeys Manual of Systematic In less than 15 years, there has been a marked increase in Bacteriology. Volume 5: The Actinobacteria. 2nd ed. New requests for DT testing of C. diphtheriae and C. ulcerans in York, NY: Springer; 2012. p. 245-89. Canada. This increase could be due to an enhanced ability 3. Bernard K. The genus corynebacterium and other medically to identify these agents using MALDI-TOF systems. Ongoing relevant coryneform-like bacteria. J Clin Microbiol 2012 monitoring of these bacterial strains will help to assess whether Oct;50(10):3152–8. DOI PubMed the increase in NML referrals is due solely to increased precision 4. Public Health Agency of Canada. Vaccine-Preventable of diagnosis or whether these are emerging cutaneous Diseases—Diphtheria: For Health Professionals. PHAC; pathogens. 2018. https://www.canada.ca/en/public-health/services/ immunization/vaccine-preventable-diseases/diphtheria/ health-professionals.html Authors’ statement 5. Public Health Agency of Canada. National case definition: Diphtheria. Surveillance. PHAC; 2018. KAB wrote the manuscript and reviewed the data https://www.canada.ca/en/public-health/services/ ALP, TB and DW performed all technical assays and reviewed the immunization/vaccine-preventable-diseases/diphtheria/ manuscript health-professionals/national-case-definition.html 6. Lowe CF, Bernard KA, Romney MG. Cutaneous diphtheria Conflict of interest in the urban poor population of Vancouver, British Columbia, Canada: a 10-year review. J Clin Microbiol 2011 None. Jul;49(7):2664–6. DOI PubMed 7. Griffith J, Bozio CH, Poel AJ, Fitzpatrick K, DeBolt CA, Cassiday P, Kenyon C, Smelser C, Vagnone PS, Culbreath K, Acknowledgements Acosta AM. Imported Toxin-Producing Cutaneous Diphtheria - Minnesota, Washington, and New Mexico, 2015–2018. We gratefully acknowledge the following centres for sending MMWR Morb Mortal Wkly Rep 2019 Mar;68(12):281–4. study strains to the National Microbiology Laboratory: DOI PubMed Dr. L Hoang, BC Centre for Disease Control, Vancouver, British 8. Meinel DM, Kuehl R, Zbinden R, Boskova V, Garzoni C, Fadini Columbia; Dr. G Tyrell and S Gee, Provincial Laboratory, D, Dolina M, Blümel B, Weibel T, Tschudin-Sutter S, Widmer Edmonton, Alberta; R Kitzel, R Romanow Provincial Laboratory, AF, Bielicki JA, Dierig A, Heininger U, Konrad R, Berger Regina, Saskatchewan; Dr. P Van Caeseele and Dr. D Alexander, A, Hinic V, Goldenberger D, Blaich A, Stadler T, Battegay Cadham Laboratory, Winnipeg, Manitoba; Dr. J Kus, Public M, Sing A, Egli A. Outbreak investigation for toxigenic Health Ontario, Toronto, Ontario (ON); Dr. H Dick, Ideex Corynebacterium diphtheriae wound infections in refugees Laboratories, Richmond Hill, ON; Dr. MC Domingo, Laboratoire from Northeast Africa and Syria in Switzerland and Germany de Santé Publique du Québec, Sainte-Anne‑de‑Bellevue, by whole genome sequencing. Clin Microbiol Infect 2016 Dec;22(12):1003.e1–8. DOI PubMed Quebec; Staff, the Moncton Hospital, Moncton, New Brunswick (NB); Staff, Hôpital Georges-Dumont, Moncton, 9. Reynolds GE, Saunders H, Matson A, O’Kane F, Roberts NB; M Saab, Atlantic Veterinary Hospital, Charlottetown, SA, Singh SK, Voss LM, Kiedrzynski T. Public health action following an outbreak of toxigenic cutaneous diphtheria in an Prince Edward Island. We thank current and former students Auckland refugee resettlement centre. Commun Dis Intell Q A Vachon, D Kielich and J Ung for technical services provided. Rep 2016 Dec;40(4):E475–81. PubMed

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